This application claims priority from Norwegian patent application 1999 5978, filed on Dec. 6, 1999.
The present invention relates to a receptor binding conjugate which consists of an antibody, a radionuclide and folate or folate derivative, wherein or not the conjugate possesses dual binding ability. The present invention also relates to a method and a kit to prepare, as well as a method to use such conjugates. Furthermore, the use of a conjugate according to the present invention to prepare a pharmaceutical solution is disclosed.
The use of folate and folate derivatives to target tumours expressing folate binding protein (FBP), a glycosyl-phosphatidyl-inositol-linked cell membrane protein involved in cellular uptake of oxidised folates via endocytosis, has attracted attention among researchers [Kranz et al, 1996; Reddy et al, 1998; Shinoda et al, 1998; Trippet et al, 1999]. As several types of human cancer cells have been shown to overexpress FBP, this receptor may be a possible target for delivery of therapeutic radioisotopes conjugated with folate. It hag been shown that various types of low-molecular weight folate-chelate-radionuclide conjugates could be used to target cells expressing FBP both in vitro and in vivo [Mathias et al, 1998]. However, the pharmacokinetics may not favour small molecules as they generally are rapidly eliminated from the body, and thereby exposing folate receptors in the kidneys to higher concentration of radiopharmaceuticals. Furthermore, the tumour uptake would be limited, as the blood concentration in the target tissues decreases rapidly.
In a previous study, Shinoda et al. (1998) evaluated folate conjugated bovine serum albumin (BSA) labelled with the radionuclide, indium-111, and found that there was a significant difference in pharmacokinetics and biodistribution of non-folate compared to the folate labelled BSA. A high-liver uptake and rapid blood clearance indicated that the folate labelled version of 111In-BSA was not particularly suitable for radionuclide delivery to tumour cells expressing folate binding protein.
The combination of folate-antibody-radionuclide has to our knowledge not been presented before, although the combination of folate and antibody has been evaluated for other uses Kranz, et al. (1996) conjugated folate to an anti-effector cell antibody with the intent of (1) binding the folate-antibody to folate binding protein on the target cells and (2) affecting lysis of the targeted calls due to the antigen binding portion of the antibody interacted with an effector cell. However, the FBP receptor system has not yet been successfully used to target radionuclides to cancer cells.
In order to target radionuclides to the FBP containing tumours in locoregional settings in particular, it may be advantageous to use larger carriers that do not diffuse too rapidly from the region in which they are injected. Monoclonal antibodies have been raised against the FBP (Campbell et al., 1991), but the problem with monoclonal antibodies is that they usually contains whole protein or peptide sequences from non-human species, and are therefore to a various degree immunogenic when used in humans. This may hamper the use of monoclonal antibodies in protocols requiring repetitive injections because of the appearance of human anti-mouse-antibodies or human anti-chimeric-antibodies, which causes immunocomplexation and unfavourable biodistribution of radioimmunoconjugates (Bruland et al., 1995; Meredith et al., 1993). Also, monoclonal antibodies and their fragments are usually only just monovalent or bivalent (i.e. not multivalent) with respect to antigen combining sites.
It is therefore the object of the present invention to provide an effective receptor selective tumour targeting agent (conjugate) prepared from a carrier molecule (i.e. an antibody), a radionuclide and folate which is chemically linked to the carrier. The conjugate should also simultaneously retain a significant antigen combining ability for the original antigen; i.e. the conjugate possesses dual binding ability. Furthermore, it is an object of the present invention to provide a method to prepare and use such conjugates. These objects have been obtained by the present invention, characterised by the enclosed claims.
The present invention relates to a new class of folate derivatives (conjugates), as well as a method to prepare and use such conjugates. The conjugate consists of (1) an antibody or antibodies preferably of IgG or IgM class. or fragments or constructs (e.g. minibody) thereof, (2) a radionuclide and (3) folate. According to the present invention, the conjugate contains only human proteins/peptides and may have increased avidity, due to the possibility of conjugating multiple folates per antibody, and thereby increasing the probability of binding the conjugate to the target cells. The antibody which is used is either an inert antibody, or a monoclonal antibody with affinity towards a tumour associated antigen. In the first case, a conjugate with FBP affinity alone is constructed, whereas in the second case a conjugate with potentially dual reseptor affinity is made, as the antigen retains a significant antigen combining ability for the original antigen, as well as binding ability for folate to FBP. The principal advantage of dual receptor affinity is that the abundance of target cells without at least one receptor for the conjugate will be reduced, increasing the chances of a succesfull targeting. To our knowledge we are the first to propose the use of folate-antibody-radionuclide to deliver radiation to tumour cells.
The present invention will now be described in more detail, with reference to figures and examples.